A variety of nuclear magnetic resonance methods will be employed to investigate a series of selected heme proteins for the purpose of understanding the mechanisms of control of function for hemoglobins. This program will emphasize, but is not restricted to, the exploration and development of the information content of hyperfine shifts in paramagnetic forms of hemoproteins in terms of the detailed molecular/electronic structure in the heme cavity. The ultimate goal is to be able to determine from NMR data the structural consequences of single-point mutations which result in impaired function in Hb A. The cornerstone of our program is the continued unique utilization of selectively isotope-labeled hemes to locate and assign 1H, 2H and 13C heme resonances and the development of indirect NMR methodology for definitively assigning potentially functionally relevant amino acid side chains in the heme cavity. The systems to be studied are selected on the basis of data available to facilitate interpretation of NMR spectral parameters in terms of the difference in structure that can be correlated with difference in function. Central to our study are a series of myoglobins and allosteric monomeric (Chironomus) and dimeric (Chironomus, E. inequivalvis) hemoglobins which serve as simplified models for Hb A. The assigned peaks will be used to quantitatively describe the hyperfine shift origins in terms of detailed structure in low-spin ferric proteins, to develop empirical correlations with structure of the heme cavity in deoxy myoglobins and hemoglobins, and to develop a variety of general NMR probes for structure-function relationships in hemoproteins. The information content and scope of applicability of NMR detection of myoglobin in intact muscle will be explored. The phenomenon of equilibrium heme orientational disorder and the factors influencing protein-heme peripheral contacts will be elucidated in both native and reconstituted hemoproteins, with emphasis on the influence of heme orientation on intra-subunit interactions in dimeric and tetrameric hemoglobins. The oxygen affinity of individual disorder components will be determined. The mechanism of reaction of apo-proteins with hemes and the role of heme orientationally disordered intermediates in hemoprotein reconstitution and biosynthesis will be characterized in a variety of myoglobins and hemoglobins. The structural probes developed for the model myoglobins will be applied towards elucidating the molecular mechanism of allostery in monomeric, dimeric and tetrameric hemoglobins.